Clostridium difficile-associated disease (CDAD) is an illness characterized by damage to the intestine and a marked inflammatory response. Until recently the disease was generally seen in hospitalized patients or those on antibiotics for prolonged periods. However, the disease is becoming increasingly prevalent, particularly among otherwise healthy individuals in the community. Coincident with its increased prevalence, the disease is becoming more severe with well documented increases in morbidity and mortality. Current antibiotic treatment of CDAD is often ineffective, leading to the search for alternate therapeutic approaches. The cardinal virulence factors of C. difficile are a pair of closely related toxins A and B (also known as TcdA and TcdB). Recent in vivo studies demonstrate TcdB to be the most essential to C. difficile virulence. Both enzymes transfer glucose from UDP-glucose (UDPG) to host GTPase proteins, resulting in their inactivation and subsequent cell death. Both enzymes also possess a glycohydrolase activity that cleaves UDPG to free UDP and glucose. There are currently no therapies that specifically target these toxins. Since the intestinal damage and clinical disease caused by C. difficile is predominantly due to toxin B, our goal is to identify specific inhibitors of this toxin. We have cloned and expressed the catalytic domain of TcdB, developed two assays to detect its activity, and have adapted both assays to 384-well format. Before proceeding with high throughput screening, these assays will be compared in a pilot HTS. Confirmatory and secondary assays will be optimized for the evaluation of TcdB inhibitors identified by HTS. In the second year of this proposal we plan to enter our assay into the Molecular Libraries Screening Probes Network (MLSPN). Longer term goals are to structurally optimize toxin inhibitors in vitro and in vivo models of C. difficile associate disease. We anticipate that the most promising of these will be developed as potential therapies against CDAD.